Methylation of chromatin recruits various regulatory proteins to govern gene expression and represents the most common epigenetic regulator. To understand this common mechanism for the modulation of gene expression it is important to study the interaction between human methylation enzymes and their chromatin targets (histone proteins). Currently, the study of HMT enzymes relies largely on peptide-based substrates, which can be engineered to include epigenetic methylation marks among other post-translational modifications (PTMs). Alternatively, histone proteins can be used as substrates and are typically derived from cultured cells or recombinant sources. However, neither peptide nor histone approaches are likely to sufficiently recapitulate the in vivo structure of polynucleosome chromatin, rendering questionable their translational value (i.e., ability to reveal molecular and physiological behavior in vitro that closely resembles in vivo phenomena). Alternative substrates for methyltransferase assays include nucleosomes purified from biological materials (e.g., HeLa cells, avian erythrocytes) but present their own translational issues by using immortalized or non- human cell types. Also, these sources comprise chromatin with varying methylation states. Employing substrates with pure and known methylation marks is crucial, as some enzymes recognize singly methylated residues toward catalyzing di- and tri-methylation events, with each state potentially exerting unique effects on gene expression. To address the significant gap between the current tools available and the need for substrates with high translational value, EpiCypher has pioneered and optimized methods for installing PTMs in recombinant histones to form designer nucleosomes. We have focused initial development of designer nucleosomes to fill the unmet and urgent need for human methyltransferase (HMT) assays. HMTs regulate the most common epigenetic mark (methylation) and represent the target for several marketed therapeutics. Given the association of aberrant HMT regulation with human disease, nucleosome-based HMT assays offer a transformational opportunity for drug discovery. These reagents also hold the potential to become the key substrate in screening for new therapeutic interventions. Therefore, while the overall goal of the proposal is to develop HMT assays with translational value, we also recognize the need for high-throughput implementation for future therapeutic screening. To accomplish this goal we will leverage Perkin Elmer's AlphaScreen(r) platform. The resulting screening platform, referred to here as AlphaNuc(r), will use biotin-streptavidin conjugation of nucleosomes to magnetic beads as a substrate for HMT activity assays. The project leverages our prior experience in the synthesis of recombinant, PTM-modified nucleosomes, as well as our relationship with an expert in this field (Dr. Alex Ruthenburg, University of Chicago).